In 2015, this project provided state-of-the-art research technologies for NIAID's intramural infectious diseases, allergy, and immunology research programs. The new technologies are developed, validated, and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the NIAID intramural program. The technologies supported include flow cytometry, confocal microscopy, DNA microarray, protein separation, mass spectrometry, peptide synthesis, protein sequencing, and macromolecular X-ray crystallography. Many of these technologies are used in high containment laboratories critical to the Institute's infectious diseases and biodefense research agenda. In addition to technology development, the RTB provides advanced training in all aspects of the technologies in the Branch's portfolio. Flow Cytometry The Flow Cytometry Section (FCS) provides the DIR with application-specific flow cytometric technologies, including instrumentation for high-speed cell sorting and multi-color analysis (up to 13-color sorting, up to 14-color analysis, BSL-3 sorting and analysis, multispectral imaging cytometry, and multiplex bead array assays). In addition, the FCS provides training, consultation, method development, and analysis. A staff with extensive flow cytometry experience and access to eight cell sorters and eleven analyzers, offers cutting-edge applications to NIAID investigators. The major goals are to provide access to state-of-the art technologies, to help design and run experiments, to facilitate data interpretation and to provide results that are of consistent high quality. The Section also provides a cost- and time-efficient method of obtaining fluorescent-labeled custom antibodies. Resources available include hybridoma expansion; antibody purification from ascites, antisera, and hybridoma supernatant; and coupling of purified antibodies and proteins to various fluorochromes. Biological Imaging The Biological Imaging Section (BIS) offers instrumentation and expertise in advanced light microscopy imaging technologies including confocal, fluorescence, video microscopy, and post-collection quantification and deconvolution. Additionally, the Section is a resource for Stimulated Emission Depletion (STED) microscopy, multiphoton confocal microscopy (intravital imaging), sample preparation, long time-lapse imaging, the NIAID Imaris licenses, and poster printing. Staff collaborates with investigators in experimental design, instrument operation, and increasingly sophisticated image processing routines to best utilize the power of these cutting-edge technologies. Investigators participate in the collection of their data, either as direct microscope operators or by selecting cells of interest, so that the staff can then collect the data. BIS advises on the most suitable instrument, appropriate labels, sample preparation, and best sampling regimens. The Section anticipates new directions for imaging needs of DIR and comes well-equipped and knowledgeable in those areas. The staff advises on the correct use of the most appropriate software analysis modules, as well as the best format for displaying the data. Frequently, the Section works with investigators to prepare images for publication including still images, video sequences, 3D reconstructions and time-lapse animations. Protein Chemistry The Protein Chemistry Section develops project specific applications in the fields of peptide synthesis, N-terminal (Edman) sequencing, protein separation, assay development, and mass spectrometry. The RTB designs qualitative and quantitative physical-chemical and biochemical methods of detection and analysis that are customized to meet the specific research needs of DIR investigators. The Branch is also active in formulating and executing purification strategies for various types of molecules. This can also include customized small scale sample preparation and enrichment strategies for analysis by mass spectrometry. Another major activity of the RTB involves the development of more efficient and effective methods of sample preparation with a strong orientation towards mass spectroscopy to facilitate protein identification work. The Branch collaborates with DIR investigators to develop and validate protocols for protein separation and analysis for specific research programs of interest to the intramural research program. The Mass Spectrometry Facility provides support in the planning and execution of mass spectrometry (MS) projects and cooperates with other labs within the RTB to support analysis and characterization of peptides, proteins, and small bio-molecules. The facility offers all facets of mass spectrometry projects including experimental design, separation of biomolecules, sample preparation, data collection and analysis/interpretation of data. The MS facility focuses on analyzing proteins and peptides, and characterizes small molecules upon request. Genomic Technologies The Genomic Technologies Section (GTS) provides state-of-the-art microarray and Next Generation Sequencing applications, and other massively parallel genomic technologies to intramural investigators. Applications are developed in close collaboration with DIR investigators. The section develops project-specific research applications for microarray platforms including custom chip design, experimental design, sample processing and chip processing. Statistical analysis, data management, and data mining solutions are available as well, including from experimental concept to publication and public data submission. In addition to managing high-end instrumentation, computers, and protocols involved in modern genome-wide assays, the GTS offers statistical analysis and bioinformatics support as well. This support involves collaborating with the investigators in both experimental design and data analysis to best utilize the power of these applications, as well as to convey a better understanding of the technology and experimental results. The Section maintains instrumentation and resources for Agilent, Illumina, and other microarray platforms, Illumina (Solexa) high throughput sequencing, commercial and custom bioinformatics software, and scientific computing. The section provides bioinformatics support for all of the offered technologies and applications. Structural Biology The Structural Biology Section (SBS) provides specialized techniques, equipment, and scientific expertise that enable DIR scientists to obtain structural and biophysical information for macromolecules. Through close collaboration with DIR researchers, the SBS determines X-ray crystal structures of proteins and protein-ligand complexes. X-ray crystallography requires expertise in producing large amounts of high-purity correctly folded proteins, preparing diffraction-quality crystals, and determining crystal structures by X-ray methods. The SBS provides consulting, produces proteins, performs biochemical and biophysical techniques, and determines structures of proteins and other macromolecules that are central to the infectious disease and immunology research programs of the DIR. The Section emphasizes providing training in biochemical, biophysical, and structural methods to investigators who can then make use of structurally-based ideas and techniques in their own laboratories.